ABSTRACT Disorders of sexual development (DSD), ranging in severity from genital abnormalities to complete sex reversal, are among the most common human birth defects with incidence rates reaching almost 3%. Although causative alterations in key genes controlling gonad development have been identified, the majority of DSD cases remain unexplained. To improve the diagnosis, we screened 116 children born with idiopathic DSD using a clinically validated array-based comparative genomic hybridization platform. 8951 controls without urogenital defects were used to compare with our cohort of affected patients. Clinically relevant imbalances were found in 21.5% of the analyzed patients. Most anomalies (74.2%) evaded detection by the routinely ordered karyotype and were scattered across the genome in gene-enriched subtelomeric loci. Among these defects, confirmed de novo duplication and deletion events were noted on 1p36.33, 9p24.3 and 19q12-q13.11 for ambiguous genitalia, 10p14 and Xq28 for cryptorchidism and 12p13 and 16p11.2 for hypospadias. These variants were significantly associated with genitourinary defects (P = 6.08×10(-12)). The causality of defects observed in 5p15.3, 9p24.3, 22q12.1 and Xq28 was supported by the presence of overlapping chromosomal rearrangements in several unrelated patients. In addition to known gonad determining genes including SRY and DMRT1, novel candidate genes such as FGFR2, KANK1, ADCY2 and ZEB2 were encompassed. The identification of risk germline rearrangements for urogenital birth defects may impact diagnosis and genetic counseling and contribute to the elucidation of the molecular mechanisms underlying the pathogenesis of human sexual development.

[Show abstract][Hide abstract]ABSTRACT: Mammalian sex determination is the unique process whereby a single organ, the bipotential gonad, undergoes a developmental switch that promotes its differentiation into either a testis or an ovary. Disruptions of this complex genetic process during human development can manifest as disorders of sex development (DSDs). Sex development can be divided into two distinct processes: sex determination, in which the bipotential gonads form either testes or ovaries, and sex differentiation, in which the fully formed testes or ovaries secrete local and hormonal factors to drive differentiation of internal and external genitals, as well as extragonadal tissues such as the brain. DSDs can arise from a number of genetic lesions, which manifest as a spectrum of gonadal (gonadal dysgenesis to ovotestis) and genital (mild hypospadias or clitoromegaly to ambiguous genitalia) phenotypes. The physical attributes and medical implications associated with DSDs confront families of affected newborns with decisions, such as gender of rearing or genital surgery, and additional concerns, such as uncertainty over the child's psychosexual development and personal wishes later in life. In this Review, we discuss the underlying genetics of human sex determination and focus on emerging data, genetic classification of DSDs and other considerations that surround gender development and identity in individuals with DSDs.

[Show abstract][Hide abstract]ABSTRACT: Despite the fact that genitourinary defects are among the most common birth defects in newborns, little is known about their etiology. Here we analyzed children born with congenital genitourinary tract masculinization disorders by array-comparative genomic hybridization, which revealed in 1.35% of cases the presence of de novo copy number gains at Xq28 encompassing the VAMP7 gene, which encodes a vesicle-trafficking protein that is part of the SNARE complex. Transgenic mice carrying a bacterial artificial chromosome encoding human VAMP7 mimicked the defective urogenital traits observed in boys with masculinization disorders such as cryptorchidism, urethral defects and hypospadias. Transgenic mice also exhibited reduced penile length, focal spermatogenic anomalies, diminished sperm motility and subfertility. VAMP7 colocalized with estrogen receptor α (ESR1) in the presence of its cognate ligand, 17β-estradiol. Elevated levels of VAMP7 markedly intensified ESR1-potentiated transcriptional activity by increasing ESR1 protein cellular content upon ligand stimulation and upregulated the expression of estrogen-responsive genes including ATF3, CYR61 and CTGF, all of which have been implicated in human hypospadias. Hence, increased gene dosage of VAMP7, and thus higher expression levels of its protein product, enhances estrogen receptor action in male genitourinary tissues, affects the virilization of the reproductive tract and results in genitourinary birth defects in humans.

[Show abstract][Hide abstract]ABSTRACT: Normal development of the genitourinary (GU) tract is a complex process that frequently goes awry. In male children the most frequent congenital GU anomalies are cryptorchidism (1-4%), hypospadias (1%) and micropenis (0.35%). Bladder exstrophy and epispadias complex (BEEC) (1∶47000) occurs less frequently but significantly impacts patients' lives. Array comparative genomic hybridization (aCGH) identified seven individuals with overlapping deletions in the 2p15 region (66.0 kb-5.6 Mb). Six of these patients have GU defects, while the remaining patient has no GU defect. These deletions encompass the transcription factor OTX1. Subjects 2-7 had large de novo CNVs (2.39-6.31 Mb) and exhibited features similar to those associated with the 2p15p16.1 and 2p15p14 microdeletion syndromes, including developmental delay, short stature, and variable GU defects. Subject-1 with BEEC had the smallest deletion (66 kb), which deleted only one copy of OTX1. Otx1-null mice have seizures, prepubescent transient growth retardation and gonadal defects. Two subjects have short stature, two have seizures, and six have GU defects, mainly affecting the external genitalia. The presence of GU defects in six patients in our cohort and eight of thirteen patients reported with deletions within 2p14p16.1 (two with deletion of OTX1) suggest that genes in 2p15 are important for GU development. Genitalia defects in these patients could result from the effect of OTX1 on pituitary hormone secretion or on the regulation of SHH signaling, which is crucial for development of the bladder and genitalia.

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Identification of De Novo Copy Number VariantsAssociated with Human Disorders of SexualDevelopmentMounia Tannour-Louet1*, Shuo Han2, Sean T. Corbett1, Jean-Francois Louet2, Svetlana Yatsenko3,Lindsay Meyers3, Chad A. Shaw3, Sung-Hae L. Kang3, Sau Wai Cheung3, Dolores J. Lamb1,2*1Scott Department of Urology, Baylor College of Medicine, Houston, Texas, United States of America, 2Department of Molecular and Cellular Biology, Baylor College ofMedicine, Houston, Texas, United States of America, 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States ofAmericaAbstractDisorders of sexual development (DSD), ranging in severity from genital abnormalities to complete sex reversal, are amongthe most common human birth defects with incidence rates reaching almost 3%. Although causative alterations in keygenes controlling gonad development have been identified, the majority of DSD cases remain unexplained. To improve thediagnosis, we screened 116 children born with idiopathic DSD using a clinically validated array-based comparative genomichybridization platform. 8951 controls without urogenital defects were used to compare with our cohort of affected patients.Clinically relevant imbalances were found in 21.5% of the analyzed patients. Most anomalies (74.2%) evaded detection bythe routinely ordered karyotype and were scattered across the genome in gene-enriched subtelomeric loci. Among thesedefects, confirmed de novo duplication and deletion events were noted on 1p36.33, 9p24.3 and 19q12-q13.11 forambiguous genitalia, 10p14 and Xq28 for cryptorchidism and 12p13 and 16p11.2 for hypospadias. These variants weresignificantly associated with genitourinary defects (P=6.08610212). The causality of defects observed in 5p15.3, 9p24.3,22q12.1 and Xq28 was supported by the presence of overlapping chromosomal rearrangements in several unrelatedpatients. In addition to known gonad determining genes including SRY and DMRT1, novel candidate genes such as FGFR2,KANK1, ADCY2 and ZEB2 were encompassed. The identification of risk germline rearrangements for urogenital birth defectsmay impact diagnosis and genetic counseling and contribute to the elucidation of the molecular mechanisms underlyingthe pathogenesis of human sexual development.Citation: Tannour-Louet M, Han S, Corbett ST, Louet J-F, Yatsenko S, et al. (2010) Identification of De Novo Copy Number Variants Associated with HumanDisorders of Sexual Development. PLoS ONE 5(10): e15392. doi:10.1371/journal.pone.0015392Editor: Syed A. Aziz, Health Canada, CanadaReceived August 17, 2010; Accepted September 1, 2010; Published October 26, 2010Copyright: ? 2010 Tannour-Louet, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: This work was supported by Grants 1R01DK078121 from the National Institute of Kidney and Digestive Diseases to DJL, the American UrologicalAssociation Foundation (SC and DJL), and by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01HD36289) to DJL. Thefunders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: dlamb@bcm.edu (DJL); mlouet@bcm.edu (MTL)IntroductionThe acquisition of a sexual phenotype depends on criticalembryonic steps, which initially commit the bipotential gonad toeither a testis or an ovary and direct normal morphogenesis ofexternal genitalia. Disruption of these developmental processesoccurs frequently in humans as reflected by the high prevalence innewborns of disorders of sexual development (DSD) ranging inseverity from genital abnormalities to complete sex reversal.Failure of testis descent or cryptorchidism is found in 2% of full-term males [1]. Hypospadias or defects in the growth and closureof the external genitalia affect nearly 1 in 125 live male births [2].Genital phenotypes that are not clearly male or female areestimated to occur in about 1 of 2000 to 4500 babies [3]. Despitetheir incidence, the molecular basis underlying the pathology ofcongenital genitourinary (GU) defects is surprisingly poorlyunderstood. Fetal exposure to environmental toxicants [4,5], aswell as point mutations in a small subset of genes (see for review[6,7]) can affect human urogenital tract development, but theseknown causes do not account for all of the large number of GUbirth defects. Interestingly, as referenced in the Online database ofMendelian Inheritance in Man (http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim), a significant number of these urogenitalinborn errors are associated with major congenital malformationsor multiple minor anomalies, a trait that is highly suggestive of acausative chromosomal abnormality. However, routine cytogenet-ic methods had led to earlier reports of low rates of structuraldefects associated with disorders of sexual development [8,9].The finding that several common syndromes (including mentalretardation, developmental delay and autism) are caused byspecific submicroscopic chromosomal rearrangements, opened upnew avenues for dissecting complex human phenotypes [10,11].The development of comparative genomic hybridization (CGH)into a microarray format allowed the identification and diagnosisof cryptic deletions or duplications of genomic regions that wereonce invisible using traditional cytogenetic methods, includingkaryotype analysis and fluorescence in situ hybridization (FISH).Several of these subtle rearrangements occur in regions flanked bylow-copy repeats and likely result from non-allelic homologousrecombination between different copies of these repeats duringPLoS ONE | www.plosone.org1 October 2010 | Volume 5 | Issue 10 | e15392

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meiosis. Such submicroscopic imbalances lead to copy numberchanges of DNA segments and can influence gene expressionlevels by directly disrupting genes or regulatory sequences,creating fusion genes or altering gene dosage. These structuralchromosomal defects can cause disease as occurs in themicrodeletion and microduplication syndromes [12,13,14,15,16]or confer risk of complex disorders [17,18].Cryptic chromosomal rearrangements are involved in theetiology of human reproductive disorders since Y chromosomemicrodeletions are associated with human male infertility. Basedon this, we tested the hypothesis that submicroscopic chromosomalalterations, too small to be detected by routine cytogeneticmethods, may exist in patients with human disorders of sexualdevelopment. We studied probands presenting with hypospadias,cryptorchidism and ambiguous genitalia, the most commongenital defects seen in pediatric urology clinics. We comparedthe resolution of clinical detection of such cryptic abnormalities bymicroarray-based chromosomal screening and by the routinelyused karyotype. We further analyzed the contribution of thesestructural anomalies to the observed GU phenotypes by studyingtheir association with the genital traits, as well as their inheritanceand their recurrence. For the first time, findings revealed thepresence of frequent microdeletions and microduplications in thegenome of children born with urogenital disorders and establishedde novo germline rearrangements as significant risk factors fordevelopmental defects of human urogenital tract.MethodsEthics Statement, Human Subjects and Sample CollectionThis study was approved by the Institutional Review BoardCommittee at the Baylor College of Medicine, Houston TX.Probands affected with unexplained syndromic and non-syndro-mic congenital genitourinary disorders including hypospadias,cryptorchidism or ambiguous genitalia were enrolled throughTexas Children’s Hospital and Ben Taub General Hospital,Houston TX. Known causes of these birth defects such asanomalies in the synthesis of testosterone or adrenal steroidhormones or exogenous modifiers were ruled out after examina-tion by pediatric urologists or neonatologists. Written informedconsents were obtained for infant/child subjects and from theirparents. Blood was collected from the children during surgery forcorrection of the GU defects. Parents provided saliva specimens.Based on the novel CMA findings, additional cases were thenidentified through an existing database from Kleberg CytogeneticsLaboratory (Baylor College of Medicine, Houston TX). Theseadditional probands were referred patients, mostly presenting withexternal genital ambiguity with or without subclinical phenotypes.Clinical indications at the time of the referral were taken fromcrude clinical comments on laboratory requisitions.CGH based Microarray Analysis (CMA)High molecular weight genomic DNA isolated from peripheralblood or saliva was submitted for chromosomal microarrayanalysis (CMA) to the Clinical Cytogenetics Laboratory at BaylorCollege of Medicine. CMA is a clinically validated targeted CGHarray that covers over 150 distinct human clinically relevantchromosomal loci [19,20]. Three different versions of CMA havebeen used depending on the time of sample submission. Theversions 5 and 6 contain 3 to 10 BAC/PAC clones per genomicdisorder specific locus and subtelomeric regions, with CMA V.5.0consisting of 853 BAC clones and CMA V.6.1 consisting of 1475BAC clones with inclusion of 1 clone per band at 650 cytogeneticbanding resolution. The newer version Oligo V.6 uses 42,640oligonucleotides of 60 base pairs with an average of 20 to 40oligonucleotides corresponding to each CMA V.6.1 BAC clonegenomic locus. Importantly, data acquired from the arrayplatforms CMA V.6.1 and CMA Oligo V6 were shown to bequalitatively comparable, allowing for cross comparison analysis[21]. One unique DNA reference served as a control for CMAanalysis and was from a pregnancy-proven fertile, gender-matchedindividual without any familial history of congenital genitourinarydefects. CMA procedures and data analyses were performed aspreviously described [19,20,21].All data are MIAME compliant and have been deposited in aMIAME compliant database.Interpretation of CNV significanceClinically significant CNVs included detection of well-charac-terized deletion/duplication syndromes,.3 Mb in size or cytogenetically visible, and de novo deletions orduplications ,1 Mb. Imbalances that were not associated withwell-characterized human syndromes were defined as ‘‘likelybenign’’ when the variant was well documented to occur in thenormal population on the basis of public databases (http://projects.tcag.ca/variation) or internal lab experience whichincludes analysis of about 16,000 individuals. In cases in whichnon-polymorphic defects were ,1 Mb in size and parentalsamples were unavailable, variants were considered to be CNVsof uncertain clinical significance. Maternally inherited copychanges were included in this latter category as the rearrange-ments may be causative without necessarily translating into similarabnormal GU traits in the female genitourinary tract.deletion/duplicationCNV confirmationFISH analysis was used to validate selected CMA findings.150 Kb in size, using the standard clinical cytogeneticslaboratory protocol [22]. Briefly, BAC clone DNA probes werelabeled directly with Spectrum Orange-dUTP or SpectrumGreen-dUTP using a commercially available kit (Abbot Molecu-lar/Vysis). At least 10 metaphase and/or 50 interphase cells werescored for each hybridization. A control probe, labeled in theopposite color, was included in the same hybridization in order toconfirm that cells were diploid (ploidy control).Quantitative TaqMan copy number variant (CNV) assays(Applied Biosystems) were used as an alternate secondary con-firmation to FISH analysis. All reactions with TaqMan CNVassays were performed in triplicate using the FAM dye label-basedassay for the target of interest and the VIC dye label-basedTaqMan CNV RNaseP for the internal controls. The targets werecustom designed in the areas where most significant changes in theprobes were detected. QPCR was performed with 20 ng gDNAaccording to the manufacturer’s protocol in an Applied BiosystemsOne Step Plus Real-Time PCR System using the default universalcycling conditions. Relative quantitation analysis was done toestimate copy number for each sample by using the Copy CallerSoftware V1.0 (Applied Biosystems).Concurrent G-banding KaryotypeMetaphase preparations from PHA-stimulated patient lympho-cyte cultures followed a standard protocol to obtain chromosomesat $600–50-band level [23]. Briefly, after being cultured for,72 hours in RPMI 1640 with 20% fetal bovine serum,lymphocytes were synchronized by the addition of thymidine for24 hours of culture, followed by the addition of ethidium bromideand colcemid for the last 45 minutes and 25 minutes of culture,respectively. The cells were treated for 20 minutes with 0.075 MKCl and were fixed in 3:1 methanol–acetic acid prior to staining.Structural Variation and Urogenital DevelopmentPLoS ONE | www.plosone.org2October 2010 | Volume 5 | Issue 10 | e15392

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The chromosomes were GTG-banded, and $20 chromosomalspreads were examined.Statistical AnalysisTo analyze the frequency of de novo copy number changes inaffected GU patients compared to unaffected GU individuals (non-GU controls), two-tailed Fisher’s exact test was performed andstatistical significance determined using SPSS software. Since (i) denovo events were only observed in GU patients run on CMA V.6.1and CMA Oligo V6 (n=90 out of the total of 116 analyzed GUchildren) and (ii) data acquired from the array platforms CMAV.6.1 and CMA Oligo V6 were shown to be qualitativelycomparable [21], comparison of frequencies was done for casesand non-GU controls run only on CMA V.6.1 and CMA OligoV6. P values were also determined for each of the spontaneousevents to evaluate their association with the GU phenotype ascompared to their specific occurrence in individuals without GUdefects (n=8951). Significance threshold was set at P=5.061022.ResultsDetection of Non-Polymorphic Imbalances Leading toVariations in Copy Number in the Genome of ChildrenBorn With Disorders of Sex DevelopmentHigh molecular weight genomic DNA was isolated fromperipheral blood of 116 children presenting with unexplainedcases of disorders of sexual development ranging in severity frompenile growth or testicular descent anomalies to genitaliaambiguity or complete sex reversal. Since the primary goal ofthis study was to improve the diagnosis of these urogenital defectsand rapidly translate the findings to the clinic, DNA was analyzedusing an established CGH microarray platform available forclinical diagnosis (chromosome microarray assay, CMA) [19,20].This targeted array specifically assesses relative copy numberchanges for over 150 human clinically relevant chromosomal loci.One unique DNA reference served as a control for CMA analysisand was from a pregnancy-proven fertile, gender-matchedindividual without any familial history of congenital genitourinarydefects. Copy number variants (CNVs) were classified based ontheir clinical significance (see Methods). Basically, CNV pathoge-nicity depended on whether a given CNV overlapped with aknown genomic syndrome that includes urogenital defects amongthe clinical features, or was present in a patient with similarphenotype, was not a copy number variant in healthy individuals,arose de novo (but not exclusively) and contained at least one gene.Chromosomal imbalances were detected in 37 (31.9%) of the116 patients analyzed (Table 1). When compared to thepolymorphisms documented in public CNV databases (http://projects.tcag.ca/variation) or based on internal lab experience,which included analysis of about 16,000 individuals, 83.8% (31 of37) of these defects were non-polymorphic (Table 1). FISH orqPCR secondarily validated these structural variants. Theyspanned the genome and affected sex chromosomes, as well asautosomal regions (Figure 1 and Tables 2–5), an observationconsistent with the fact that male sexual development is governedby genes not restricted to the Y chromosome [7]. Most of thegenomic rearrangements (25 of 37 i.e. 72.9%) were clinicallysignificant copy number variants (Table 1 and Tables 2–4).Detection rates of these clinically relevant aberrations were slightlycomparable between the three studied genital conditions (25% forambiguous genitalia, 17.2% for hypospadias and 18.5% forcryptorchidism) (Table 1). Interestingly, these genomic abnor-malities were noted in patients presenting with both syndromic, aswell as non-syndromic genitourinary disorders (Table 1), with noTable 1. Submicroscopic imbalances revealed by CMA screening of children affected with syndromic and non-syndromicdisorders of sex development.No Aberration Chromosomal AberrationsTotalRate of Detectionof Non-Polymorphic CNV (%)Rate of Detectionof ClinicallySignificant CNV(%)NormalBenignCNVNon-Polymorphic CNVClinicallySignificantUCSAmbiguous GenitaliaIsolated cases 213 111 36 33.327.8Associated with other anomalies 18141 2420.8 16.0Total 394 152 60 28.325.0HypospadiasIsolated cases12032 1729.417.7Associated with other anomalies81211225.016.7Total201532927.6 17.2CryptorchidismIsolated cases1502017 11.811.8Associated with other anomalies513110 40.030.0Total20151 2722.218.5Total79625611626.721.5Footnote: UCS: Uncertain Clinical Significance.doi:10.1371/journal.pone.0015392.t001Structural Variation and Urogenital DevelopmentPLoS ONE | www.plosone.org3October 2010 | Volume 5 | Issue 10 | e15392

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statistically significant difference in their respective detection rates.From a clinical perspective, this latter observation stresses theimportance of screening children presenting with isolatedhypospadias or cryptorchidism, who usually are not referred forgenetic testing.Regardless of the GU condition, the size of CMA detectedanomalies ranged from 50 kilobases to 57.4 Mb with an averagedefect size of 5.5 Mb, which is at the limit of the resolution ofroutine karyotype (Tables 2–5). Importantly, none of theimbalances smaller than 5 Mb, which represent about 70% ofthe identified CMA defects, were detected by concurrent high-resolution karyotype analysis (Tables 2–5). For the cases ofimbalances larger than 5 Mb, CMA analysis had proven toprovide a better definition of the structural defect than thekaryotype (patients 6, 9, 12, 13, 18- see Tables 2–5). Moreover,most of the detected imbalances (70.6%) were subtelomeric defects(Tables 2–5), which are known to be difficult to characterize by G-banding due to their location in the distal G-negative stainingregions of the chromosomes. The most illustrative finding was seenin patient 15 with a referring diagnosis of ambiguous genitalia(Table 2). A large deletion of 57.4 Mb spanning the subtelomeric10q26 was only suspected by karyotype, but was successfullydetected by CMA analysis. Interestingly, this deletion encom-passed FGFR2, a particularly noteworthy candidate gene in light ofrecent studies in rodents that found evidence for its role in testisformation and male sex determination [24,25]. While genevariants of FGFR2 may influence the risk of hypospadias inhumans [26], conditional inactivation of FGFR2 in mouse modelsresulted in blockade of the XY-specific gonad growth anddisruption of testis differentiation, leading to a male-to-femalesex reversal phenotype. The characterization of FGFR2 as a sex-determining gene in the mouse suggests that the CMA detectedhuman haploinsufficiency of FGFR2 is a strong candidate defectunderlying the phenotype of abnormal male genital developmentin patient 15.Interestingly, in three unrelated CMA screened patients (27, 28and 30; Table 2), a low-level mosaic state became apparent afterCMA screening and retrospective analysis of the karyotype.Figure 1. Comprehensive map of non-polymorphic copy number changes detected by CMA in patients with disorders of sexdevelopment. On the right, CMA detected imbalances were shown for each clinical condition (asterisks). To gain insight into the genomicdistribution of the identified imbalances, all published single gene mutations associated with cryptorchidism (blue), hypospadias (green) andambiguous genitalia (red) were reviewed and indicated on the left side of the chromosomes. References are available upon request.doi:10.1371/journal.pone.0015392.g001Structural Variation and Urogenital DevelopmentPLoS ONE | www.plosone.org4 October 2010 | Volume 5 | Issue 10 | e15392

Interphase FISH performed on blood smears in which multiplecell lineages coexist, was requested after CMA testing, to verifymosaicism in these children. The conventional karyotype wasnormal since it examined only the cell population of stimulated Tlymphocytes. The fact that CMA analysis performed on DNAfrom uncultured blood cells was able to improve the detection oflow level mosaicism missed by cytogenetic analysis, is of significantclinical importance, especially for the diagnosis of genitalambiguity.Strong Association of De Novo Copy Variants withHuman Disorders of Sex DevelopmentThe inheritance of the FISH-confirmed CMA defects wasinvestigated by CMA testing. Parental samples were not availablefor all patients, leading to an underestimation of the clinicallysignificant abnormalities in the present evaluation. De novooccurrences were noted for: (i) deletions in 1p36.33, 9p23p24and 19q12-q13.11 for probands presenting with a referringdiagnosis of ambiguous genitalia; (ii) duplications in 10p14 andXq28 for cryptorchid children; (iii) and deletions in 12p13.31-p13.2 and 16p11.2 for patients with hypospadias (Table 6).Importantly, these de novo copy number changes were found to bemore frequent in patients with congenital genitourinary defectsthan in control individuals without GU abnormalities (28 out of8951), withanassociationthat(P=6.08610212; Fisher’s exact test) (Tables 6 and 7).To a lesser extent, imbalances inherited from a phenotypicallynormal maternal parent were also considered since the rearrange-ments may be causative without necessarily translating into similarabnormal GU traits in the female genitourinary tract. Thus,maternally inherited copy changes were considered as of unclearclinical significance and noted as: (i) deletion in 7p22.1 forambiguous genitalia; (ii) duplications in 4q35.2 and 5p15.31 foris statisticallysignificanthypospadias; (iii) duplication in 5p15.2 and in the androgenreceptor insensitivity region, Xq12 for cryptorchidism (Table 5).Hence, the present analysis shed light on spontaneouschromosomal rearrangements affecting novel and unsuspectedgene-enriched regions that have potential to contribute to thepathogenesis of human genital development.Unrelated Patients Presenting with Similar Genital TraitsShared Common Affected LociThe causal link of the CMA defects to the GU phenotype wasfurther strengthened by the fact that common overlapped lociwere shared by unrelated probands having similar genitaldefects. Spontaneous deletion of the 9p23p24 region was foundin patients 9, 12, and 13, all with gonadal dysgenesis (Table 2).A minimal common region of overlap included 260 kb of9p24.3 (Figure 2A). This smallest reported sex-reversing 9pdeletion appears therefore as a hotspot for the pathogenesis ofsex determination. It encompasses KANK1, DOCK8 and DMRTgenes. The testis specific DMRT1 is worthy of mention, since itencodes a protein-sharing domain homology with the doublesex(Dsx) of Drosophila and Mab3 of Caenorhabditis. Both of these genesare crucial for the normal sexual development of theseorganisms. Genetic inactivation of DMRT1 demonstrated itsrequirement for the development of the male gonad [27], butdid not lead to sex reversal in XY mice, suggesting theinvolvement of additional interacting factors in order tophenocopy the human phenotype. One of such gene candidatescould be KANK1 since it is highly expressed in the mouseembryonic genital tract (http://www.genepaint.org) and is ableto physically interact and regulate the subcellular localization ofbeta catenin whose activation in normal XY mice has beenshown to disrupt the male program and result in male-to-femalesex-reversal [28,29].Table 6. De novo clinically relevant copy number changes detected in patients presenting with disorders of sex development(DSD).ID Locus DSD diagnosisCNVStartPositionSize(Mb)Genes% InNon-GU% InGUP valueKaryotypeInh17 12p13.31p13.2HypospadiasLoss7,987,9842.306650.011.11 1.96102246,XY dn2016p11.2HypospadiasLoss 29,729,9700.131 100.071.11 5.76102246,XY dn34Xq28HypospadiasGain154,703,3210.158102.22 9.96102346,XY dn33Xq28 CryptorchidismGain 154,703,3210.15810 2.229.96102346,XY dn1610p14CryptorchidismGain 12,011,8060.06430.021.11 2.961022n/a dn1 1p36.33 Ambiguous Genitalia Loss799,476 1.257 65 0.171.11 1.46102146,XX dn12 9p23p24.3Ambiguous Genitalia(Gonadal dysgenesis)Loss 356,238 9.774 660.042.221.46102346,XY,der(9)del(9)(p23)dup(9)(p23p12)dn139p24.1-pter Ambiguous Genitalia(Gonadal dysgenesis)Loss1 6.785580.042.221.46102346,XY,der(9)del(9)(p24pter)dup(9)(p23p12)dn9 9p24.3Ambiguous Genitalia(Gonadal Dysgenesis)Loss356,238 0.2594 0.042.221.46102346,XY,del(9)(p23)dn2219q12q13.11 Ambiguous GenitaliaLoss 33,828,5275.638 4901.11 9.96102346,XYdnFootnotes:Minimal size of the spontaneous aberrations (Mb) and the number of the encompassing HGNC (Hugo Gene Nomenclature Committee) genes (G) (NCBI Build v35.1)were indicated.P values were based on two-tailed Fisher’s exact test comparing the frequency of each spontaneous event in cases versus controls. Significance threshold was set atP=5.061022.Abbreviations: Inh: Inheritance, dn: de novo.doi:10.1371/journal.pone.0015392.t006Structural Variation and Urogenital DevelopmentPLoS ONE | www.plosone.org9 October 2010 | Volume 5 | Issue 10 | e15392

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In addition to the 9p region, the locus Yp11.31 was an expectedrecurrent hit in patients with ambiguous genitalia, since itencompasses SRY, the testis-determining gene (Table 2).Unique Similar Loci were Abnormal in PatientsPresenting with Different Urogenital TraitsIdentical unique loci were affected in patients presenting distinctgenital phenotypic traits, which suggest that structural perturba-tions within these segments may alter master regulator(s) ofmultiple processes of human sexual development. Indeed, a largedeletion of the subtelomeric cytoband 5p15 detected in the patient6 with genital ambiguity overlapped a region duplicated in patient5 with hypospadias (Figure 2B). The fact that the shared genomicinterval spanned the locus of the Cri-du-Chat syndrome, whichincludes hypospadias among its various clinical features, providessupport for a causative link of the 5p15 defect to the GUphenotype. The encompassed gene, ADCY2, appeared as an idealcandidate controlling genitourinary development since it presentsa high and specific expression pattern in the testis as well as in thedeveloping genital tract (http://www.genepaint.org). Moreover,the ADCY2 encoding protein regulates the intracellular levels ofcyclic AMP, a crucial second messenger in major regulatorypathways involved in the biogenesis of the genital system such asSonic Hedgehog signaling.The DiGeorge syndrome critical region 22q11.2 was also foundduplicated in patients with ambiguous genitalia, while its deletionwas seen in patients presenting with GU abnormalities inassociation with Smith-Lemli-Opitz syndrome or Wilms tumor(Table 2). This region may have a dosage sensitive gene(s) thatplays a role in the development of the genitourinary system inhumans.In parallel, alterations occurring on 2q22 and Xq28 loci werefound in patients with cryptochidism and hypospadias (Tables3–4). These genomic regions may contain candidate genes thatregulate a common protagonist(s) or pathway(s) controlling bothtesticular descent and formation of the male urethra. The deletion2q22 in patient 2 presenting with cryptorchidism and hypospadiaswas in the region associated with Mowat-Wilson syndrome(MWS). Among anomalies frequently observed in MWS areurogenital anomalies including hypospadias and undescendedtestis. MWS is a genetic condition caused by heterozygousmutations or deletions of ZEB2 (zinc finger E-box bindinghomeobox 2 gene), a protein that interacts with a receptor-mediated, activated full-length SMAD. ZEB2 is strongly expressedin the developing murine genital tract (http://www.genepaint.org). Knockout mice models of ZEB2 presented reproductivesystem defects ([30], http://www.informatics.jax.org). Moreover,ZEB2 has been shown to modulate Wnt signaling, a criticalpathway for the development of the genital tract [31]. Hence,ZEB2 appears as a potential candidate involved in the maleurogenital development.Taken together, our findings highlight for the first time thepresence of previously unrecognized chromosomal imbalances aspotential genetic risks factors in disorders of sexual developmentand illustrate how a microarray-based technology provides apowerful alternative to traditional cytogenetic and gene-mappingapproaches for discovering contributing factors in disease ofcomplex etiology.DiscussionThe development of male reproductive system is a complexprocess controlled by delicate networks that specify sex-specificdifferentiation, organogenesis and endocrine function. Thefragility of these regulatory cascades is illustrated by the highprevalence of genitourinary defects in newborns. These inbornurogenital anomalies present difficult challenges for the parentsand the physicians, as care of these children is complicated bysurgical, psychological, social and sexual concerns. The goldstandard for genetic diagnosis remains a karyotype analysis and anendocrine profile but findings in intersex cases are not alwaysinformative. Indeed, only a small portion of these developmentalaberrations can be attributed to defects in the synthesis oftestosterone or adrenal steroid hormones, receptor alterations,exogenous modifiers or obvious numerical and structural chro-mosomal alterations, such as Klinefelter syndrome. The underly-ing causes of the majority of ‘‘idiopathic’’ cases remain to bediscovered. In this study, the use of a clinically validatedmicroarray (CMA) revealed the existence of cryptic imbalancesstrongly associated with defects of urogenital development orrecurrently found in patients with DSD. These chromosomalaberrations were mostly too small to be detected by the routinelyordered karyotype, which has a limited resolution of 5–10 Mb,depending on the quality of chromosome preparations. Many ofthese genomic anomalies went also largely undetected becausethey were located in subtelomeric loci, which are notoriouslydifficult to characterize by G-banding. Moreover, mild or isolatedcases of hypospadias and cryptorchid patients are usually notreferred for genetic testing, while this study proved that this subsetof patients harbored structural variation that may convey defectiveurogenital traits.Most of the detected chromosomal aberrations encompassedone to a few hundred genes including known gonad-determininggenes (SRY and DMRT1) as well as novel candidate genes such asFGFR2, KANK1, ADCY2 and ZEB2. Changes in dosage orstructure of genes within the affected DNA segments might leadto haploinsufficiency or altered transcription profiles, which maydisturb the intricate fine-tuned network of genes controlling thehuman genital development. Clinically relevant examples of genedosage alterations have already been documented for factorscontrolling mammalian sex development. For instance, deletion ofthe sex-determining gene WNT4 is responsible for the masculin-ization of XX mouse pups, while its duplication and overexpres-sion in humans leads to XY sex reversal [32,33]. Duplications oflarge segments of DNA containing DAX1 or SOX9 also cause sexreversal [34,35]. Thus, our findings contribute in a coherentmanner to strengthen the emerging concept that sex determina-tion and differentiation are dosage sensitive at multiple steps oftheir pathways. In addition to dosage effects, imbalances may leadTable 7. De novo CMA detected events are more enriched inGU patients than in individuals without urogenitalabnormalities.Sample GroupTotalPatients*Patients withde novoevents RatioP valueGenitourinary Defects90 100.116.08610212Non-Genitourinary Defects8951 28 0.003Footnotes: Two-tailed Fisher’s exact test was used to evaluate the association ofCMA detected de novo events with urogenital defects. *: GU cases (n=90 out ofthe total of 116 analyzed GU children) and non GU controls (n=8951) run onlyon CMA V.6.1 and CMA Oligo V6, since de novo events were specificallyobserved in GU patients screened with these two qualitatively comparableplatforms (n=10; see Table 6); [21]. See Statistical Analysis in Methods fordetails.doi:10.1371/journal.pone.0015392.t007Structural Variation and Urogenital DevelopmentPLoS ONE | www.plosone.org 10October 2010 | Volume 5 | Issue 10 | e15392